Genome engineering is an emerging field in which targeted genome modifications are made for biotechnological and therapeutic applications. Site specific rare cutting endonucleases are a crucial tool for genome engineering, as they are required to create DNA double strand breaks at specific genomic targets. Resolution of nuclease-induced DNA breaks by endogenous DNA repair pathways results in high efficiency genome editing at the desired target. Precision Genome Engineering has developed proprietary methods for generation and isolation of rare cutting endonucleases based on the use of the I-AniI LAGLIDADG homing endonuclease as a scaffold. This phase I SBIR will support the application of this approach to generate one or more novel LAGLIDADG nucleases capable of cleaving the human glucocortocoid receptor gene. The novel GR1-targeted nucleases will be evaluated for their capacity to generate glucocorticoid receptor (GR1)-deficient T-cells. Generation of GR1-deficient T-cells that recognize specific viral epitopes represents a novel and promising approach to therapy of chronic viral infections in individuals undergoing immunosuppression with corticosteroids. Thus, the nucleases emerging from this SBIR will be directly translatable to clinical applications with commercial potential. For this purpose, Precision Genome Engineering has established a collaboration with an academic laboratory experienced in cell therapy to obtain pre-clinical data in human T- cells, as part of a planned phase II application that would continue the development of work initiated in this phase I proposal towards commercial applications. PUBLIC HEALTH RELEVANCE: This project will support development of an engineered site specific nuclease capable of cleaving the human glucocorticoid receptor gene. This protein or refined derivatives will be applicable to generation of steroid- resistant T-cells for use in therapy of chronic viral infections in immunosuppressed patients.